Oxidative damage of erythrocytes infected with Plasmodium falciparum

Summary The extent of reduced glutathione, activity of glutathione peroxidase, amount of membrane lipid peroxidation products, and the extent of hemoglobin release from host erythrocytes during in vitroPlasmodium falciparum growth was studied. Highly synchronized parasite cultures were studied to examine the alterations caused by different growth stages of the parasite. There was a moderate increase in the reduced glutathione content as the parasite matured, which was significant only in schizontrich erythrocyte lysates (p<0.05) whereas the activity of glutathione peroxidase was significantly low in all the parasitized red blood cells (ring-infected RBC,p<0.005; trophozoite- and schizont-infected RBC,p<0.001). The lipid peroxidation product, malonyldialdehyde, of the host red cells increased gradually to more than fourfold in schizont-rich cells as compared with normal erythrocytes (p<0.001). The hemoglobin release from cultured cells was significantly higher in all parasitized red cell cultures as well as in uninfected cells kept in in vitro, as compared with normal erythrocytes. The consequence of such changes induced by the malarial parasites in the host red cells in the pathogenesis of erythrocyte destruction and anemia ofP. falciparum malaria is discussed.     

Studies on the Fetal Hemoglobin in the Persistent High Hb-F Anomaly

Three generations of a Negro family having herditary persistence of fetalhemoglobin in conjunction with the hemoglobins A and S were studied.

Genetic studies did not exclude the possibility that this anomaly is allelicwith the genes responsible for the hemoglobins A, S, and C.

Structural investigations of the isolated abnormal hemoglobin fraction offered evidence that this component is identical with the fetal hemoglobin ofthe newborn child.

No striking clinical or hematologic abnormalities were found in membersof the family who had high percentages of fetal hemoglobin in conjunctionwith either Hb-A or Hb-S. The per cent of Hb-A₂ in the persistent high Hb-Fcarriers was found slightly below normal, while the total amount of circulatinghemoglobins was increased. Previous reports of the complete absence ofHb-A in cases heterozygous for Hb-S as well as for the persistent high Hb-Fanomaly have been confirmed by using refined technics available for hemoglobin analyses.

The fetal hemoglobin was distributed almost equally in all erythrocytes ofindividuals having this inherited anomaly. In this respect, the S-F abnormalitydiffers from homozygous sickle-cell disease in which a mixed population ofred blood cells, some with and most without Hb-F, has been demonstrated. Itis suggested that the presence of some Hb-F in all erythrocytes of the individual with the S-F condition is the factor that protects the cells fromsickling and from destruction.

The distribution of fetal hemoglobin in the red blood cells of persistent highHb-F carriers was also different from that found in patients with thalassemiamajor and thalassemia-Hb-E disease. Two distinct cell populations, one withand the other without fetal hemoglobin, were found to be present in theblood of the thalessemia patients.

Submitted on April 21, 1961 Accepted on June 18, 1961     

Autoerythrocyte sensitization; a form of purpura producing painful bruising following autosensitization to red blood cells in certain women

Four patients with purpura who manifested an unusual response to bruisingwere studied. This response was characterized by the development of an areaof painful ecchymosis at the site of trauma followed by progressive erythemaand edema. This unusual tissue response was seen only in women. The variousfeatures of the cases suggested an autosensitization by the patients to theirown blood.

Special studies utilizing skin testing procedures indicated an abnormal tissueresponse of sensitivity to red blood cells. The factor responsible was presentin the red cell stroma and was not associated with the hemoglobin.

The clinical manifestations and possible therapy are discussed.

This syndrome may represent another example of autosensitization such ashas been speculated for lupus erythematosus, some forms of acquired hemolyticanemia and of thrombocytopenic purpura, and for an increasing number of disease states.

Submitted on October 5, 1954 Accepted on February 7, 1955     

Fast Hemoglobin in Lead Poisoning

1. An electrophoretically fast hemoglobin was found in approximately 40per cent of preschool children with elevated blood lead levels.

2. Fast hemoglobin was found more often in lead-poisoned patients withhypochromic anemia than in patients with normochromic red cells.

3. Fast hemoglobin differed from hemoglobins produced in vitro by incubation with chromate or oxidized glutathione. It had electrophoretic propertiessimilar to that found in a few patients receiving tolbutamide.

4. Fast hemoglobin could not be differentiated from normal hemoglobin A₃by any technic utilized.

5. Both lead and A₃ hemoglobins were heterogeneous molecular species.

6. The mechanisms leading to the production of hemoglobin A₃ and leadhemoglobin remain unknown.

Submitted on October 7, 1965 Accepted on December 10, 1965     

Sickle Cell--Hemoglobin C Disease: Quantitative Determination of Iron Kinetics and Hemoglobin Synthesis

Quantitative determinations of iron kinetics and hemoglobin synthesis weremade on five patients with sickle cell-hemoglobin C disease. The anemia wasmild in all patients but one who had the hemoglobin of 10.1 Gm. per cent.All patients were in a steady state during the period of this study.

The ferrokinetic determinations demonstrated a hemolytic process in allcases. The mean erythrocyte life span in these patients was 18, 20, 29, 46and 56 days respectively (normal range, 110-130 days). The hemoglobinsynthesis was increased in all. Reduced to terms of daily hemoglobin production per liter of blood, the values were 2.2 Gm., 2.9 Gm., 3.6 Gm., 5.8 Gm.and 6.6 Gm. The latter figure represents a five fold increase over the normalmean value of daily hemoglobin synthesis of 1.3 Gm. (normal range, 1.0 to1.6 Gm. of hemoglobin per liter of blood per day).

The results of the in vivo organ counts demonstrated a significant degreeof sequestration of red blood cells in the spleen of two patients. The questionof advisability of splenectomy in such patients was discussed.

Accepted on December 3, 1962     

The anemia of the Di Guglielmo syndrome

The physiopathology of the anemia of the Di Guglielmo syndrome (erythremic myelosis) was studied in 11 patients with the acute and chronic varieties of the disease. Ferrokinetic studies were performed in three additionalpatients.

1. The anemia was normochromic and macrocytic; in contrast to the meancorpuscular volume, which was elevated, the mean corpuscular hemoglobinwas often normal. In several patients the mean corpuscular hemoglobin concentration was slightly lower than normal, suggesting slight hypochromia.

2. Reticulocytes were often increased but bore no relationship to the degree of the anemia nor to the shortening of the red cell life span. The reticulocyte count is an unreliable index of blood production in this disease.

3. The degree of erythroblastemia was highly variable. No direct correlation existed between the degree of erythroblastemia and the acuteness of thedisease, nor was there any relationship between the degree of erythroblastemiaand either the degree of anemia or the degree of erythrocytic destruction.

4. The bone marrow showed striking erythroblastic hyperplasia. This wasusually of the megaloblastic type. Primitive erythroblasts (erythrogones) wereconspicuous. The erythroblastic hyperplasia was out of proportion to the relatively minor reticulocytosis or the relatively slight diminution in red cellsurvival.

5. The nucleated red cells of the marrow showed variable numbers ofmegaloblasts and megaloblastoid forms, suggesting the presence of a vitaminB₁₂ deficiency (pernicious anemia). However, the vitamin B₁₂ concentrationof the serum was elevated, and there was no response to the administrationof vitamin B₁₂ or folic acid.

6. Varying numbers of erythroblasts in the bone marrow and in the peripheral blood showed periodic acid-Schiff (PAS)-positive granules in the cytoplasm. No chemical abnormalities of hemoglobin could be detected either bythe method of paper electrophoresis or by the alkali denaturation test.

7. Diminished red cell survival was present in most cases, but it was of arelatively slight degree. It was due to an "intracorpuscular" defect of the redcells.

8. The often great increase in fecal urobilinogen output as compared witha relatively minor rate of red cell destruction suggests "heme pigment diversion" or increased destruction of precursor red cells, as in pernicious anemia,where the same phenomenon has been observed.

9. The great increase in the number of erythroid cells in the bone marrowand the increased rate of iron turnover as compared with the relatively minorincrease in red cell destruction and iron utilization point to an "ineffective"type of erythropoiesis. The high degree of "ineffective erythropoiesis" seenin this disease may be characteristic of the neoplastic proliferation of the redcell series.

10. In conclusion, the anemia of the Di Guglielmo syndrome is due to acombined disturbance: (1) an "ineffective" type of erythropoiesis of markeddegree, perhaps due to an acquired (neoplastic) defect in the uptake or utilization of B₁₂ by the erythroblasts and (2) increased hemolysis resulting fromthe increased destruction of defective red cells.

Submitted on June 11, 1958 Accepted on August 13, 1958     

Studies on the pancytopenia of kala-azar

1. The peripheral blood changes in uncomplicated kala-azar are those ofpancytopenia; namely, anemia, leukopenia and thrombocytopenia. The red bloodcell morphology is normal and there is very little evidence of increased erythrogenicactivity. The leukopenia is due to a reduction in all types of cells, especiallyneutrophils.

2. When the disease is complicated by other infections the anemia is moresevere and anisocytosis, poikilocytosis, and polychromatophilia may appearand normoblasts may occasionally be seen in the peripheral blood. Leukocytosismay develop, the leukopenia may persist or the syndrome of agranulocytosis mayintervene.

3. As the duration of the disease increases, the spleen tends to become largerand the anemia, leukopenia and thrombocytopenia become progressively moresevere. Leukopenia generally appears first, followed by anemia and finally thrombocytopenia. The degree of leukopenia, anemia and thrombocytopenia followclosely the degree of splenic enlargement.

4. The bone marrow in kala-azar is hyperplastic and infiltrated by reticulo-endothelial cells. In spite of this there appears to be an abundance of blood formingtissue, especially erythropoietic tissue.

5. Differential cell studies on preparations of sternal marrow reveal a markedreduction in the polymorphonuclear neutrophils and eosinophils. Myeloblasts,promyelocytes, neutrophilic myelocytes and metamyelocytes are present in approximately normal proportions as are the lymphocytes and monocytes. Plasmacells are somewhat increased. Erythroid cells, especially polychromatic normoblasts are numerous and the leukocyte-erythroid ratio is altered, more than thenormal proportion of normoblasts being found. Megakaryocytes are present innormal or slightly reduced numbers. Staining abnormalities are noted in thesecells and there is a striking reduction in platelet production.

6. During effective anti-leishmanial therapy parasitized reticulo-endothelialcells disappear and the percentage of reticulo-endothelial cells gradually diminishes as the polymorphonuclear neutrophils increase. There is a significantincrease in the eosinophilic cells. Lymphocytes become more numerous and theplasma cells diminish in number. Nucleated red cells become less numerous andthe leukocyte-erythroid ratio returns to normal. At the same time the proportionof orthochromatic normoblasts to polychromatic normoblasts increases. Therelative number of megakaryocytes increases and platelet formation from themegakaryocytes is accelerated even beyond the normal. Huge groups of plateletsare frequently seen in the marrow smears. A rise in platelets in the peripheral bloodtakes place late, after there has been a significant rise in hemoglobin and leukocytes. The three cellular elements are restored to normal in the peripheral blood inthe same order as their reduction from normal.

7. Evidence is presented which contradicts the view that the pancytopenia isdue to a crowding out of the bone marrow by reticulo-endothelial cells.

8. Certain similarities between the hematologic changes in this disease andthose accompanying the hypersplenic syndromes are noted.

     

Hereditary nonspherocytic hemolytic disease; a study of a singular familial hemolytic syndrome

Extensive studies were performed on four cases from three unrelated kindredswith a familial hemolytic syndrome not associated with any significant red cellanomaly (hereditary nonspherocytic hemolytic disease). These cases were compared with similar ones already reported in the literature.

1. Hereditary nonspherocytic hemolytic disease appears to be transmitted asa Mendelian dominant. Frequently the gene responsible for the condition seemsto have low expressivity. In some cases, the hereditary mechanism may be dueto inheritance of a recessive gene from each parent. The basic erythrocytic defectresponsible for the condition is unknown. In view of various clinical and hematologic findings, it is likely that hereditary nonspherocytic hemolytic disease maybe a group of diseases involving more than one mechanism.

2. All criteria of hemolytic anemia (erythroid hyperplasia of the bone marrow,reticulocytosis, hyperbilirubinemia, increased fecal urobilinogen, rapid turnoverof tracer iron in the plasma) were satisfied.

3. Red cell survival time studies revealed an intraerythrocytic defect with amean life span of twelve to seventeen days. Normal red cells transfused into thepatients under study survived normally. Anemia was normochromic and normocytic or macrocytic; it varied from mild to severe.

4. Osmotic and mechanical fragility of the red cells was normal. Osmotic andmechanical fragility tests after incubation at 37 C. for 24 hours in some showed amild increase compared with normal controls. Autohemolysis of incubated oxalated blood was not marked and varied from case to case.

5. The electrophoretic mobility of hemoglobin from the patients was that ofnormal adult hemoglobin. Small increases of fetal hemoglobin were seen in several cases.

6. In contrast to the histologic findings in hereditary spherocytosis the splenicpulp was not congested, but hemosiderin deposits were heavy. Liver biopsy specimens showed deposits of hemosiderin in parenchymal and Kupffer cells.

7. Splenectomy did not arrest the hemolytic process. Mild improvement was seenin one case. In most cases the operation is of no value.

8. Diagnostic difficulties may be encountered with mild cases of hereditaryspherocytosis. Examination of rouleaux in fresh blood and an osmotic fragilitytest in 0.65 per cent sodium chloride after incubation usually establishes the differential diagnosis. The condition may present clinically as hemolytic disease ofthe newborn and must be differentiated from erythroblastosis due to Rh or otherblood group incompatibilities. Other hereditary hemolytic diseases such assickle cell anemia, Cooley’s anemia, hereditary spherocytosis, and hereditaryhemolytic elliptocytosis are easily ruled out by their typical clinical and hematologic manifestations. When a family study is negative or cannot be done, a redcell survival time determination may be necessary to rule out acquired hemolyticanemia with a negative Coombs test. Some cases that have been diagnosed asconstitutional hyperbilirubinemia (familial nonhemolytic jaundice) may actuallyrepresent mild hereditary nonspherocytic hemolytic disease.

Submitted on October 7, 1953 Accepted on November 3, 1953     

The Natural History of Iron Deficiency Induced by Phlebotomy

1. The sequence of characteristic changes of progressive iron deficiency wasdemonstrated by serial bleeding of normal volunteers and polycythemic patients.

2. After bloodletting, changes occurred in peripheral blood in the followingorder: a) fall in hemoglobin concentration; b) decreased plasma iron; c)reticulocytosis, increased MCV and MCH; d) diminution of MCV and MCH,increased total iron-binding protein; and e) decreased MCHC.

3. Characteristic changes of iron deficiency returned to prephlebotomylevels in the following sequence: a) hemoglobin concentration; b) cellularindices; c) serum iron; d) serum iron binding protein; and e) bone marrowhemosiderin, and finally the increased gastrointestinal absorption of ironreverted to normal.

4. Accelerated production of red cells continued in polycythemic patientsdespite the induction of moderate iron deficiency. Quality was sacrificed forquantity, and thereby a more profound microcytosis occurred than in normalsubjects with a similar degree of iron deficiency.

Submitted on February 16, 1962 Accepted on April 11, 1962     

Blood destruction in the polycythemia induced by hypoxia

The hemoglobin catabolism during the development and during the disappearance of polycythemia induced by hypoxia was studied by measuring the totalcirculating hemoglobin and the daily bile pigment excretion in bile-fistula dogsbefore, during, and after prolonged periods of exposure to 20,000 feet simulatedaltitude.

1. The inscreased erythropoiesis during the first weeks of altitude exposure wasaccompanied by a signiflcant increase in bile pigment output. The possible sourcesof this pigment excretion are discussed.

2. The life spans of the red cells during altitude exposure was found to be about115 days. No differences were observed in the longevity of the cells in animals atground level and at altitude.

3. The normalization of the polycythemic blood levels took place within sixto eight weeks after returns to ground level, and was achieved by the combinedeffect of a depressed erythropoiesis and of an increased blood destruction. Theincrease in red cell destruction observed under these conditions demonstratesthe existence of an "active" mechanism of blood destrunction by which the organism is able to destroy normal blood cells before their life span is exhausted. Thisincreased red cell destruction, however, accounted for only 21 to 39 per cent ofthe hemoglobin which disappeared from circulation after return to ground level.The major part of the normalization of altitude polycythemia was brought aboutby a temporary depression of erythropoiesis which was estimated to amount to30 or 40 per cent of the normal cell production in the six weeks after the discontinuation of the altitude exposure.

Submitted on September 15, 1951 Accepted on October 25, 1951     

The relationship between carbonic anhydrase activity and zinc content of erythrocytes in normal,in anemic and other pathologic conditions

A good correlation exists between zinc content and carbonic anhydrase activityof the red blood cells under all conditions studied, including anemia and polycythemia. In almost all patients with anemias other than pernicious anemia, bothzinc and carbonic anhydrase levels were lowered in parallel fashion. These changeswere proportional to decreases in hematocrit and hemoglobin levels and erythrocyte counts so that both zinc and carbonic anhydrase values per unit of RBC werein the normal range. In a few instances of anemia associated with leukemia andin one of sickle cell anemia, neither zinc content nor carbonic anhydrase activitywas decreased in proportion to the anemia; in these cases the zinc and carbonicanhydrase levels per unit of blood were both elevated to the same degree.

Patients with pernicious anemia showed no decrease in absolute values for zincand carbonic anhydrase activity in spite of marked lowering of hematocrit andhemoglobin levels and of erythrocyte count. Accordingly, both zinc concentrationand carbonic anhydrase activity per unit of blood were elevated, often to a markeddegree. These increases were parallel, varying inversely with the degree of anemia;when they regressed under treatment, both did so at the same rate.

There are no methods available for estimating carbonic anhydrase concentration;all methods now in use measure only the activity of the enzyme. It is suggestedthat zinc concentration could be used as an indicator of carbonic anhydrase contentof the red blood cells.

Note: ACKNOWLEDGMENTSDrs. Joseph C. Aub and Ira T. Nathanson were kind enough to refer several patients for study. Dr.Byrl J. Kennedy was most helpful in regard to obtaining samples of blood. The technical work wasperformed by Miss Mary Lou Roney, Betty Hickey and Marion Taylor.

     

Hookworm Anemia: Iron Metabolism and Erythrokinetics

Iron metabolism, balance of red cell production and destruction and ironabsorption from hemoglobin were determined in 11 patients with heavy hookworm infection and severe anemia.

The plasma iron, total iron binding capacity, bone marrow hemosiderinand plasma Fe⁵⁹ clearance are in agreement with the idea that the anemia associated with hookworm infection is of the iron deficiency type.

The rate of red cell production measured by the E/M ratio, absolute reticulocyte count and plasma iron turnover showed an increase to about twicenormal, while the rate of destruction estimated by the T erythrocytesurvival showed a destruction about 5 times normal. This unbalance betweenproduction and destruction could explain the severity of the anemia.

The increase of fecal urobilinogen output to twice normal was interpretedas due to the metabolism of the hemoglobin lost into the intestine rather than toan increase of hemolysis.

The estimation of fecal blood loss in the patients whose red cells weretagged with Cr⁵¹ and Fe⁵⁹, showed that the radioactivity counted with Fe⁵⁹was only about 63 per cent of the radioactivity counted with Cr⁵¹. This difference was interpreted as due to iron absorption from the hemoglobin lostinto the intestine.

The mean daily fecal excretion of iron reaches 4.7 mg. Since the ironmetabolism in these patients is in equilibrium, we have concluded that theiron loss is replaced by the iron from food; this is in addition to the 3 mg.hemoglobin iron which is reabsorbed from the blood lost into the gut.

Submitted on January 9, 1961 Accepted on April 2, 1961     

The mechanism of the anemia associated with Hodgkin''s disease

1. The survival of autotransfused Cr⁵¹-tagged erythrocytes was shortenedin a group of five patients with the anemia of Hodgkin’s disease, but since thenormal marrow is believed to be capable of compensating for destruction ratesof six to eight times greater than normal, it is felt that the increased rate ofhemolysis was not sufficient to account for the development of anemia in anyof these patients.

2. There was evidence of an increased rate of erythropoiesis in all of thepatients with Hodgkin’s disease. This was manifested by normoblastic hyperplasia of the marrow, a moderate reticulocytosis and increased plasma ironclearance rates. The shortened red cell survival times were associated withthe increased plasma iron clearance rates. However, in view of the fact thatall of the patients were anemic, it is evident that these patients were incapableof increasing the rate of red cell production sufficiently to compensate for theincreased rate of destruction.

3. Intravenously injected Fe⁵⁹ citrate was incorporated more rapidly intothe circulating red cell mass in the patients with Hodgkin’s disease than inthe normal subjects.

4. The tissue iron stores of the liver and spleen were greatly increased,but no iron was demonstrated in the marrow of a group of seven patientswith Hodgkin’s disease.

5. The plasma iron was low and the UIBC normal in a group of 12 patientswith active Hodgkin’s disease.

6. It is suggested that one of the factors which limits the ability of themarrow to produce hemoglobin in the anemia of Hodgkin’s disease may be arelative hypoferremia caused by a defect in the mobilization of iron from tissuestores.

Submitted on April 17, 1958 Accepted on February 2, 1959     

Erythropoietic (congenital) porphyria: A rare abnormality of the normoblasts

1. In 5 patients with erythropoietic (congenital) porphyria, unstained bonemarrow preparations were studied and photographed by fluorescence andabsorption microscopy. The same marrow slides were also studied after ordinarystaining.

2. Two morphologically different varieties of normoblasts were observed,which were designated as normal and abnormal cells. Normoblasts of theabnormal variety exhibited nuclear inclusion bodies containing hemoglobin.

3. Red fluorescence, indicative of porphyrin, was found only in normoblastsbelonging to the abnormal cell variety. Normal normoblasts failed to exhibit redfluorescence.

4. The red fluorescence originated predominantly from the normoblasticnucleus. The fluorescence intensity of the cytoplasm was usually very low.Polychromatophilic erythrocytes also exhibited only weak fluorescence.

5. These findings indicate the coexistence of two different lines of normoblasts.The nuclei of cells belonging to the abnormal line probably form excessiveamounts of porphyrins and may release them into the plasma in the course ofcellular maturation. It is believed that these cells carry the abnormal traitrepresenting the "inborn error of metabolism."

6. A critical review of all cases of porphyria, reported as "congenital," hasshown that on the basis of the presented data, it was possible to establish thediagnosis of erythropoietic (congenital) porphyria beyond reasonable doubtin but 34 instances. Nineteen of the patients were female, fourteen were male.In a few families, more than one case was observed in the same generation, butin no instance was the disease recognized in subsequent generations.

7. Erythropoietic porphyria is a congenital disease, entirely distinct fromhepatic photosensitive ("cutanea tarda") porphyria. No evidence has beenfound of a genetic mixture of these two forms of porphyria.

Submitted on July 8, 1954 Accepted on November 3, 1954     

Hereditary spherocytosis. I. Clinical,hematologic and genetic features in 28 cases,with particular reference to the osmotic and mechanical fragility of incubated erythrocytes

Clinical, hematologic and genetic data on 28 cases of hereditary spherocytosisare presented for the purpose of characterizing this disorder as completely aspossible. On the basis of this experience it is recommended that the followingtypical laboratory findings be sought in establishing a diagnosis in suspectedcases: (1) Presence of spherocytes or abnormally thick red cells in peripheralblood; (2) greater than normal osmotic fragility of the red cells; in cases in whichthe fragility of fresh cells is not significantly increased, determinations should bemade after sterile incubation of the blood at body temperature for 24 hours;(3) greater than normal mechanical fragility of freshly drawn red cells; (4) negative antiglobulin (Coombs) test; (5) greater than normal lysis of the red cellsduring sterile incubation at body temperature for 48 hours; and (6) presence ofsimilar abnormalities in relatives.

Abnormality of the erythrocyte persisted in all of the 11 patients in this seriesfollowed one or more years after splenectomy. An unusual case of chronic hemolytic anemia is described but not included in the numbered series because (1) bothparents were hematologically normal and (2) spherocytosis and abnormally greatosmotic and mechanical fragility and autohemolysis could not be demonstratedafter the fifth postoperative month. Classification of this case is deferred pendingfurther experience.

Demonstration in a parent, sibling or offspring of red cells showing the afore-mentioned abnormalities is necessary for an unequivocal diagnosis, but this requirement cannot always be met because relatives may not be available for examination. Moreover, when parents and/or several siblings are examined withoutpositive findings, low gene expressivity, gene mutation and illegitimacy may beconsidered as explanations. Evidence is cited to suggest the possibility of a lowdegree of penetrance or expression in some cases and to illustrate the need forstill more sensitive laboratory tests that might aid in diagnosis of the mildestforms of this disease. The lower incidence of spherocytosis in siblings of propositithan in offspring of propositi is cited as evidence bearing on the theory of genemutation in some propositi.

A simplified "qualitative" test of osmotic fragility of incubated red cells isdescribed.

Submitted on April 16, 1951 Accepted on May 10, 1951     

Characterization and Genetic Studies of Microcytic Anemia in House Mouse

Microcytic anemia, inherited as a unit autosomal recessive, is the first of thetwelve known single-gene induced hereditary anemias of the mouse whichresults entirely from alteration of individual erythrocytes rather than from reduction in number of red cells. At all ages where mk/mk individuals could berecognized, from the fifteenth day of gestation to adulthood, the erythrocytecount of mk/mk individuals was at least as high as that of normal counterparts.At all ages, the hemoglobin concentration in these small erythrocytes was alsoreduced, so that the total available hemoglobin was markedly reduced at allages. The higher than normal numbers of erythrocytes in adult microcyticmice demonstrate that the cell-producing mechanisms operate efficiently.Genetic tests have shown that the mk single-gene defect has no relation tostructure of either the -chain or the -chain of the hemoglobin molecule. Themk microcytosis and hypochromia must then result from a metabolic or structural defect independent both of the factors responsible for regulating red cellnumber and of those controlling hemoglobin structure.

This mk/mk microcytic anemia shows some similarities to human thalassemias: hypochromia, microcytosis and presence of target cells, combined withsplenomegaly, reticulocytosis, and higher than normal erythrocyte counts (likethalassemia minor). As in thalassemia, hemoglobin structure is normal, although the amount per cell is subnormal. Human hemolytic anemias also sharesome of these characteristics. The similarities of mk/mk microcytic anemia ofthe mouse to certain human anemias are sufficient to warrant its further investigation as an animal model for understanding of human hereditary disease.

Submitted on October 9, 1969 Accepted on January 15, 1970     

Macrocytosis Resulting from Early Denucleation of Erythroid Precursors

In an attempt to elucidate the mechanism of production of macrocytosis inacute anemia, we studied changes in red cell volume and hemoglobin content, the RNA level of normoblasts and reticulocytes and RNA synthesis inreticulocytes of rabbits made anemic by blood loss or phenylhydrazine administration. The results were as follows:

(1) In severe phenylhydrazine anemia, red cell volume and hemoglobincontent per cell increased to twice the normal values.

(2) The RNA level of normoblasts decreases with the maturation of thecells and reaches a minimum at the orthochromatic stage. The decrease issimilar in the normal and anemic rabbits.

(3) The RNA level of reticulocytes in the bone marrow is higher in anemicthan in normal rabbits. In general, the RNA level of reticulocytes of anemicrabbits is comparable to that of the polychromatic normoblasts, while in normal rabbits this value is comparable to that of orthochromatic normoblasts.

(4) Autoradiographs of reticulocytes incubated with H³-uridine indicatethat the increased level of reticulocyte RNA of anemic rabbits is not due tonewly synthesized RNA.

From these results, we conclude that in an "emergency" situation of erythropoietic stimulation denucleation of normoblasts occurs at the polychromaticstage of red cell maturation, with skipping of the terminal cell division toorthochromatic cells and formation of macrocytic reticulocytes and red cells.

Submitted on August 28, 1963 Accepted on April 30, 1964     

Erythrokinetic Studies in Severe Bone Marrow Failure of Diverse Etiology

1. Erythropoiesis was studied in seven patients with refractory anemia.

2. In all seven patients the total red cell volume was low and the plasmavolume elevated.

3. The serum iron was elevated and iron binding protein saturated in sixof seven patients.

4. The T for clearance plasma Fe⁵⁹ was decreased in one, normal in two,and increased in four patients.

5. The red cell iron turnover was decreased in six of seven patients.

6. Radioiron accumulated in liver and spleen.

7. Red cell life span was difficult to measure but probably shortened.

8. Androgen therapy in two cases was ineffective, and was associated witha slight effect in one patient.

Submitted on April 18, 1961 Accepted on September 14, 1961     

The effects of splenectomy on the red blood cells of the dog with particular emphasis on the reticulocyte response

In splenectomized dogs the following observations on the erythroid elementswere made:

1. There was no unusual postoperative depression of erythrocyte, hemoglobin or hematocrit values, and regeneration occurred at the same rate as inthe control dogs.

2. A significant increase in the number of circulating reticulocytes wasnoted, which persisted for a longer duration than the associated thrombocytosis. There is a suggestion that the reticulocytes circulating during regeneration were more immature than those of the control animals.

3. Varying numbers of target cells accompanied by increased osmotic resistance were present in all the splenectomized dogs. Most also developednormoblastemia, particularly during active regeneration, which persistedthereafter in some of the animals. Howell-Jolly bodies were rare.

4. No significant bone marrow changes were noted.

It is suggested that:

1. In nonsplenectomized animals, most erythrocytes released from the bonemarrow in response to blood loss are mature red cells rather than reticulocytes.

2. Postsplenectomy reticulocytosis is not dependent on a fall in hematocritand is not a reflection of hemolysis or even of increased erythropoiesis, except,perhaps, in part during regeneration from the blood loss incurred at surgery.

3. Postsplenectomy reticulocytosis and normoblastemia are unaccompaniedby alterations in the myeloid-erythroid ratio of the bone marrow and areprobably largely due to a diminution of the normal inhibition of erythroid release from the marrow, permitting their earlier entry into the circulation.

4. The aforementioned changes are due specifically to the removal of thespleen itself, rather than merely to the removal of splenic blood from theportal circulation.

Submitted on November 18, 1957 Accepted on April 12, 1958     

Survival studies of thalassemic erythrocytes transfused into donors,into subjects with thalassemia minor and into normal and splenectomized subjects

The analysis of the curves of thalassemic red cell survival studied in children suffering from thalassemia major, in subjects with thalassemia minor,in normal splenectomized subjects and in healthy persons shows:

(a) thalassemic erythrocytes show a marked shortening of life span,with a wide range of half life from case to case;

(b) thalassemic erythrocytes survive longer in patients with thalassemiamajor and minor than in normal subjects;

(c) the behavior of thalassemic erythrocyte survival curves in the anemicpatients and in the normal splenectomized cases is almost similar. On thebasis of these results, the authors emphasize that:

(1) valuable data on the seriousness of the hemolytic process inthalassemia major can be drawn only from autotransfusion studies;

(2) in both thalassemia major and minor the spleen has less hemolyticactivity on thalassemic red cells than in normal subjects;

(3) since survival curves between anemic and normal subjects differprincipally in the initial component where the most abnormal red cells aredestroyed by the normal spleen, it is probable that the spleen of Patients withthalassemia major is not so greatly active against these cells.

Submitted on November 27, 1956 Accepted on January 28, 1958